Hematopoiesis is the biological mechanism by which the cells of the hematopoietic system develop and differentiate. Hematopoiesis is believed to originate with hematopoietic stem cells in the yolk sac. This pluripotent stem cell then seeds to the liver at a gestational age of approximately 8 weeks. The liver is the first organ in which hematopoietic cells differentiate to produce cells of various lineages.
At approximately 12 weeks of gestational age, the stem cells also seed the other hematopoietic organs (i.e., spleen, bone marrow, thymus and lymphatic tissues). The bone marrow then takes over the role of the liver gradually, and by approximately 24 weeks, the bone marrow has taken over fully. Although the liver and spleen are believed to maintain some number of pluripotent stem cells, they usually only play a role in hematopoiesis beyond the gestational stage when there is a severe reduction in the content of the bone marrow. U.S. Ser. No. 517,101 describes a population of stem cells that comprise the pluripotent hematopoietic stem cell. This population can be identified on the basis of expression of certain cell surface antigens. Specifically, this population is CD34.sup.+ /CD38.sup.-. CD34 is the surface antigen common to all stem cells. See U.S. Pat. Nos. 4,965,204 and 4,714,680.
When fully developed, the human hematopoietic system is populated by cells of several different lineages. These differentiated cells may appear in bone marrow, thymus, spleen, liver, lymphatic tissue(s) and in peripheral blood. Within any specific lineage, there are a number of maturational stages. In most instances, the more immature developmental stages occur within bone marrow while the more mature and final stages of development occur in peripheral blood.
There are three major lineages: the erythroid lineage which matures into red blood cells; the myelomonocytic lineage which matures into granulocytes (including neutrophils, eosinophils and basophils) and monocytes; and the lymphold lineage which matures into B lymphocytes, T lymphocytes and NK cells. (The megakaryocytes may be considered a fourth lineage which gives rise to platelets.) Within each lineage and between each lineage, antigens are expressed differentially on the surface and in the cytoplasm of the cells in a lineage. The expression of one or more antigens and/or the intensity of expression are used to distinguish between maturational stages within a lineage and between lineages. Loken and Terstappen have published a series of papers which describe the maturational development of B lymphocytes (Blood, 70:1316, (1987)), the development of erythroid cells (Blood, 69:255, (1987)), the development of neutrophils (Leukemia, 4:657 (1990) and mono-myeloid cells (J. Anal. Cellular Pathol., 2:229 (1990)). In each of these cases, Loken and Terstappen worked backward from the most mature stage in peripheral blood and described the changes in light scattering and antigen expression on less differentiated cells. Distinct maturational stages could be assigned to differentiating populations of cells. Thus, for example, using B lymphocytes, Loken et al. defined four stages of development: "stage B IV" comprised the mature B lymphocytes, "stage B III" represented the immature B cell, "stage B II" represented a more immature pre-B cell and "stage B I" represented the earliest B lymphoid cell. Similar stages of development are described for each of the other lineages.
As noted in FIG. 8 of the first paper, Loken et al. defined antigen expression for each of the four described stages. Stage B I was defined by the relatively high expression of both CD34 and CD10. In stage B II, expression of CD34 decreased to very low levels, CD10 expression decreased slightly and levels of CD19, HLA-DP and HLA-DR increased significantly. In stages B III and B IV, expression of CD20, CD22 and CD21 also significantly increased.
B lymphocytes are an important part of the hematopoietic system in that these cells produce antibodies that form a major component of the immune system. T lymphocytes, on the other hand, have a number of functions which form another major component of the immune system. T lymphocytes help B lymphocytes make antibodies, recognize and destroy cells infected with viruses, activate phagocytes to destroy pathogens and control the level and quality of the immune response. The ability of both T and B lymphocytes to recognize the enormous variety of antigens to which they may be exposed results from recombination and gene rearrangement of the antigen receptor genes during development.
T lymphocytes, unlike B lymphocytes (and cells of most other lineages), develop and mature in the thymus. During embryonic development, the thymus is believed to be seeded by the T lymphocyte precursor cells first from the liver and then from the bone marrow. These precursor cells are believed to differentiate from a common lymphoid precursor, as yet undefined. From this common lymphold precursor, the precursors to B lymphocytes, NK cells and T lymphocytes develop.
The T lymphocyte precursors that seed the thymus are believed to be immuno-incompetent (i.e., they are unable to respond to specific antigens presented in association with MHC class II antigens and are unable to distinguish between self and foreign antigens.) Once in the thymus, the T lymphocyte precursors are believed to undergo three stages of differentiation as they transit from the cortex to the medulla and finally into the peripheral blood. During the period the cells reside in the thymus, the cells are referred to as thymocytes. Differentiation of thymocytes to mature lymphocytes is measured by the expression of certain cell surface antigens. The following antigens are expressed on thymocytes and T lymphocytes.
CD1 is an antigen of approximately 45 kD and is found on approximately three-fourths of cortical thymocytes. CD1 is involved in antigen presentation.
CD2 is antigen of approximately 50 kD that is expressed on nearly all T lymphocytes and thymocytes and on a subset of NK cells. CD2 is the E rosette receptor.
CD3 is an antigen of approximately 25-28 kD that is found on all mature T lymphocytes and on approximately three-fourths of thymocytes. CD3 has several chains and normally is found complexed with the T cell receptor. (TCR is a dimer composed of .alpha. and .beta. or .gamma. and .delta. subunits).
CD4 is an antigen of approximately 55 kD that is expressed on a major subset of mature T lymphocytes and nearly all thymocytes. This subset functions to help B lymphocytes to produce antibodies against a specific antigen.
CD5 is an antigen of approximately 67 kD and is found T lymphocytes, a distinct subset of B lymphocytes and thymocytes.
CD7 is an antigen of approximately 40 kD and is found on T lymphocytes, thymocytes and NK cells.
CD8 is an antigen complex consisting of two disulfide bonded subunits of approximately 32 and 43 kD. It is found on a major subset of mature T lymphocytes and the majority of thymocytes. This subset functions in the destruction of allogenic cells and virally infected target cells.
Leu 8 is an antigen of approximately 70-80 kD and is present on approximately three-fourths of T lymphocytes, approximately 45% of thymocytes, a subset of NK cells and most B cells. Leu 8 is involved in the homing of lymphocytes to peripheral lymph nodes and is also known as LECAM-1.
For a more detailed description of thymocytes and mature T lymphocytes, subsets thereof and their development and relationship to B lymphocytes, see Sci. Amer. Medicine, vol. 2, ch. 6, .sctn.I, Rubenstein and Federman eds. (1990), and see Leukocyte Typing IV, App. A, Oxford Univ. Press, Knapp et al. eds. (1989).
Because of the role of the T lymphocyte in the immune response, the loss or impairment of T cell function is important. Briefly, function can be lost or impaired as a result of a hereditary condition, damage to the thymus, drug treatment or other therapies and disease. These conditions are associated with a marked impairment of cell-mediated immune functions such as the ability to respond to infection. In addition, there can be an impairment of ability to fight tumors and to participate in and direct antibody mediated responses.
Hereditary conditions included PNP-deficiency, Di George's syndrome and chronic mutocutane candidasis. Disease conditions include viral infection (e.g., HIV infection which results in a destruction of CD4.sup.+ T lymphocytes), autoimmune disease (e.g., juvenile diabetes and systemic lupus erythmatosus which result in a loss of regulation of auto-antibodies) and leukemias (e.g., T-CLL and T-ALL). Drug and other treatments include immunosuppressive drugs (e.g., cyclosporine), chemotherapy and total body irradiation.
In each of these instances, repopulation, regeneration or replacement of one or more mature T lymphocyte subsets may be desired. For example, in HIV infections, repopulation of the CD4.sup.+ T lymphocyte subset is desirable. In autoimmune disease, replacement of only the T lymphocytes that attack self with properly functioning T lymphocytes is desirable. In those patients who have received immunotherapy, the addition of competent T lymphocytes is desirable to prevent relapse or opportunistic infection. In still other patients with T lymphocyte leukemias (e.g., T-ALL and T-CLL), removal of the leukemic cells followed by replacement with new T lymphocytes is desirable.
Because of the immunocompetence of mature T lymphocytes, one cannot use such mature cells from one person to repopulate or replace the T lymphocytes of another person having one of the above-mentioned conditions. Accordingly, if one is repopulate or replace the defective or impaired T lymphocytes, one must begin with immuno-incompetent cells. Further, it is more desirable to begin with the T lymphocyte precursor, as opposed to a stem cell, in order to generate only T lymphocytes and to it as rapidly as possible.
Accordingly, what is desired is a method to isolate and identify a population of T lymphocyte precursor cells. Once isolated, transplantation or transfusion of genetically altered or unaltered precursor cells can be effected.